毛竹笋中脱氧核糖核酸酶的提纯和特性

PURIFICATION AND PROPERTIES OF DEOXYRIBONUCLEASE FROM PHYLLOSTACHYS PUBESCENS SHOOTS

毛竹笋中的脱氧核糖核酸酶(DNasa)经热处理、硫酸铵沉淀、SephadexG-100和DEAE-纤维素柱层析被提纯27.2倍,SDS-聚丙烯酰胺凝胶电泳显示单一的带。这种酶的分子量为5.4×10~4,最适pH为5.0～5.2,最适温度为65℃,它对DNA、RNA和5~1-AMP三种底物都有水解活力,其活力比例为1:1:0.1.在pH4.8条件下,为了保持竹笋DNase活力需疏基化合物存在;但是在pH8.0条件下,疏基化合物对这种酶活力具有明显的抑制效应。除低浓度(1×10^(-5)M)的KH_2PO_4和NaF对酶活力分别抑制45％和42％。一价金属离子K^+、Na^+和NH_4+(10^(-2)M)对酶活力有一些刺激效应外,高浓度(1×10^(-3)M)的KH_2PO_2对竹笋DNase活力有10％～20％的刺激作用;在二价金属离子中,除Ca^(++)和Mg^(++)具有微弱刺激作用外,其它离子都有抑制效应。金属螯合剂EDTA(10^(-3)M)对竹笋DNase活力有明显抑制暗示,这种酶含有对活力所需要的金属离子。

The deoxyribonuclease (DNase) from Phyllostachys pubescebs shoots was purified by heat treatment, ammonium sulfate fractionation, Sephadex G-100 and DEAE-cellulose column chromatography. The data show that final purification obtained was 27.2 fold, photographs of SDS-polyacrylamide dise gels stained with Coomassie blue R-250 after electrophoresis of this DNase. preparation showed single band. The MW of the enzyme was 5.4×104. The enzyme gave pH optima of 5.0-5.2 and temperature optima of 65℃ . The purified enzyme preparation hydrolyzes denatured DNA and the 5'-AME at 1 : 1 : 0.1 activities. The absolute requirement for a sulfhydryl compound for maintenance of activity at pH 4. 8 and the marked inhibition of the enzyme activity by the same sulfhydryl compounds at pH 8.0 Except for KH2po4at lower concentration (1 x 10~5M) , there several ative effects on the enzymeactivity, approximately 45% and 42% inhibition and sodium fluoride of 1 x 10~3M. The addition of 10~2M one valent cation K+, Na+and NH+4 to the incubative mixture resulted in a 10%-20% stimulation of the DNase activity, Of a number cf two valent Cations tested, only ca++and Mg++produce a slightly stimulating effect on activity (5%-8% stimulation) , other cation found to exert markedly inhibiting effect on the activity includes Co++, Cu++, Zn++, Hg++, and Mn++. Metal chelating agent EDTA (10~3M) resulted in marked inhibition of the DNase activity, which implies that this DNase contains metal ions for maintained activity.